Method for the Purification of G-Csf

ABSTRACT

The present invention relates to a method for obtaining recombinant granulocyte-colony stimulating factor (G-CSF), comprising at least one cation exchange chromatography and at least one hydrophobic interaction chromatography, wherein said two chromatographic steps are immediately consecutive in optional order. In particular, the present invention relates to a method for purifying G-CSF from a mixture of G-CSF and other proteins, comprising two cation exchange chromatography steps which are conducted before and after a hydrophobic interaction chromatography, respectively.

The present invention relates to a method for producing recombinantgranulocyte colony-stimulating factor (G-CSF), comprising at least onecation exchange chromatography and at least one hydrophobic interactionchromatography, wherein said two types of chromatographies immediatelyfollow each other in arbitrary order. In particular, the presentinvention relates to a method for purifying G-CSF from a mixture ofG-CSF and other proteins, comprising two cation exchange chromatographysteps which are performed before and after a hydrophobic interactionchromatography, respectively.

G-CSF (granulocyte-colony stimulating factor) is a naturally occurringgrowth factor belonging in a broader sense to the family of cytokinesand herein to the group of colony stimulating factors. G-CSF plays adecisive role in hematopoiesis and enhances the proliferation anddifferentiation of hematopoietic precursor cells and the activation ofneutrophiles. Due to said characteristics, G-CSF has come to be used indifferent medical fields, like for example in the reconstitution ofnormal blood cell populations subsequent to chemotherapy or irradiationor for stimulating the immune response to infectious pathogens. Thus,clinically speaking, G-CSF is mainly employed in anti-tumor therapy andin particular in the treatment of neutropenia as a consequence ofchemotherapy and is furthermore used in bone marrow transplantations andin the treatment of infectious diseases.

Human G-CSF in its naturally occurring form is a glycoprotein having amolecular weight of about 20,000 Dalton and five cysteine residues. Fourof these residues form two intramolecular disulfide bridges which are ofessential importance for the activity of the protein. As G-CSF isavailable only in small amounts from its natural sources, recombinantforms of G-CSF are mainly used for producing pharmaceuticals, which canfor example be obtained by means of expression in mammalian cells likeCHO (Chinese Hamster Ovary) cells or in prokaryotic cells like E. coli.The recombinant proteins expressed in mammalian cells differ fromnaturally occurring G-CSF in that they have a different glycosylationpattern, while in the proteins expressed in E. coli which can have anadditional N-terminal methionine residue as a result of bacterialexpression, glycosylation is not present at all.

The recombinant production of G-CSF has been described in patentliterature for the first time in 1987, in WO 87/01132 A1. The firstcommercially available G-CSF preparation on the basis of recombinantG-CSF was admitted in Germany in 1991 and is produced and distributed byAmgen under the trade name Neupogen®.

While the production of G-CSF in prokaryotic cells is preferred ascompared to the production in mammalian cells, as the use of simplerexpression systems and culture conditions is possible, a frequentlyoccurring problem in the production of recombinant proteins inprokaryotic cells is, however, the formation of hardly solubleintracellular aggregates of denatured forms of the protein expressed,the so-called inclusion bodies, which partially have a secondarystructure and can be found in the cytoplasm of the bacterial cells.

The formation of said inclusion bodies leads to the necessity ofsolubilizing and renaturing the proteins subsequent to the isolation ofthe inclusion bodies by means of centrifugation at moderate speed withthe aid of suitable means in order to maintain their activeconfiguration. Herein, the competitive reaction between a transfer ofthe denatured protein into the right folding intermediate and anaggregation of several protein molecules is an essential factor limitingthe yield of renatured protein.

In the art, several patent documents deal with the aspect ofsolubilizing and renaturing the proteins obtained form inclusion bodies.In EP-A-0 719 860, for example, the isolation and purification of G-CSFincluding solubilization and refolding are described. General techniquesrelating to solubilization and renaturing of denatured proteins havebeen described in EP-A-0 512 097, EP-A-0 364 926, EP-A-0 219 874 and WO01/87925 and can furthermore be taken from scientific literature andstandard works on protein chemistry.

Subsequently, the refolded protein is purified by means ofchromatographic methods, i.e. it is separated from other proteins andfurther impurities which are present after solubilizing and renaturing.

WO 87/01132 A1 already mentioned in the above, wherein the production ofG-CSF in E. coli host cells has been described for the first time, alsodeals with chromatographic purification. Within the scope of thepurification of the recombinant G-CSF, a cation exchange chromatographyusing a CM cellulose column is described in Example 7 of WO 87/01132 A1.

In EP 0 719 860 A1, the G-CSF is purified subsequently to solubilizationand oxidation by means of Dowex in order to remove the solubilizingagent, followed by an anion exchange chromatography and a cationexchange chromatography. In EP 0 719 860 A1, CM sepharose is also usedfor the cation exchange chromatography.

In WO 03/051922 A1, a purification method for G-CSF is described,wherein a metal affinity chromatography is performed; more exactly, achromatography on immobilized metal (immobilized metal affinitychromatography, IMAC). Subsequently to the metal affinitychromatography, a cation exchange chromatography and/or a gel filtrationmay be performed according to WO 03/051922.

In WO 01/04154 A1, a method for purifying G-CSF is described, whereinfirst a hydrophobic interaction chromatography and a subsequenthydroxyapatite chromatography are conducted. Subsequently to thehydroxyapatite chromatography, a cation exchange chromatography isperformed.

It is a problem underlying the present invention to disclose a methodfor purifying biologically active recombinant human G-CSF, by means ofwhich it is possible to obtain G-CSF with satisfactory purity and yield.Herein, the method should be as simple and straightforward in conductionas possible. Desirable is a purification method that can be conductedwith as few chromatographic steps as possible in order to keep technicalcomplexity and costs on a low level and to avoid high losses of protein.

This and further problems are solved by means of the method given inclaim 1. Preferred embodiments are described in the dependent patentclaims.

It has been found that it is possible in the chromatographicpurification of renatured G-CSF by means of a cation exchangechromatography and a hydrophobic interaction chromatography to achieveacceptable purity of the recombinant biologically active G-CSF with asatisfactory yield. Purity can be further increased by means of a secondcation exchange chromatography step.

Thus, the present invention relates to a method for purifyingrecombinantly produced biologically active human G-CSF in which at leastone cation exchange chromatography and at least one hydrophobicinteraction chromatography are conducted, wherein said chromatographicsteps are performed in arbitrary order, provided that there is notperformed any other chromatographic step or any other purification stepbetween said steps. Thus, cation exchange chromatography and hydrophobicinteraction chromatography are immediately consecutive.

According to the present invention, the term “biologically active humanG-CSF” is understood to denote that G-CSF which has been purified bymeans of the method according to the present invention is capable ofenhancing the differentiation and proliferation of hematopoieticprecursor cells and of causing the activation of mature cells of thehematopoietic system. Thus, the G-CSF obtained by means of the methodaccording to the present invention is suitable for treating indicationsin case of which the administration of G-CSF is advantageous. It isunderstood, that the term “biologically active human G-CSF” alsoincludes mutants and modifications of G-CSF, whose amino acid sequenceis altered as compared to the wild type sequence, but which have asimilar biological activity as the wild type G-CSF like those, forexample, that are described in WO 01/87925 and EP 0 456 200. The sameapplies to G-CSF conjugates. Preferably, the G-CSF to be purified ishuman Met-G-CSF produced in E. coli cells.

In one embodiment of the present invention, the method for purifyingG-CSF comprises two cation exchange chromatography steps which areconducted before and after the hydrophobic interaction chromatography,respectively.

In a further embodiment of the present invention, the method comprises atangential flow filtration subsequent to the only or—in case more thanone cation exchange chromatography steps are conducted—the last cationexchange chromatography.

In a further embodiment, conducting an anion exchange chromatography isomitted in the method for purifying G-CSF.

In a further embodiment, the purification method according to thepresent invention is sufficient without gel filtration chromatography.

In a further embodiment of the present invention, conducting apreparative HPLC is omitted. The same applies to reversed phasechromatography, which is to be distinguished from the hydrophobicinteraction chromatography according to the present invention and whichis possibly also omitted in the preparation. rpHPLC is only employed foranalytical purposes.

In a further embodiment, no affinity chromatography, in particular nodye, metal or immunoglobulin affinity chromatography, is conductedwithin the scope of the method.

In a further embodiment, conducting a hydroxyapatite chromatography isomitted within the scope of the purification method.

Thus, in a preferred embodiment, the purification method according tothe present invention utilizes only two different chromatographicseparation methods, namely the method of ion exchange on the basis ofcompetitive interaction of charged ions and the method of hydrophobicinteraction, which is characterized in that the nonpolar surface regionsof a protein adsorb to the weakly hydrophobic ligands of a stationaryphase at high salt concentrations.

To be distinguished therefrom is the chromatographic separationprinciple of affinity which is based on the specific and reversibleadsorption of a molecule to an individual matrix-bound bonding partner.The hydroxyapatite chromatography, which is based on the use ofinorganic hydroxyapatite crystals, is a further separation method whichdiffers from the ion exchange chromatography in form of cation exchangechromatography and hydrophobic interaction chromatography.

Said chromatographic principles mentioned are also correspondinglydistinguished among experts (see, for example, Bioanalytik, F.Lottspeich, H. Zorbas (ed.), Heidelberg, Berlin, Germany, Spektrum Akad.Verlag 1998).

In a preferred embodiment, the chromatographic purification does notcomprise more than three chromatographic steps, in which only twodifferent chromatographic separation methods are employed.

Renatured G-CSF, which is supposed to be transferred to achieve a puritythat allows its use in the form of a pharmaceutical preparation, isemployed as starting material for chromatographic purification.

Herein, solubilizing and refolding the protein can be conductedaccording to the methods known in the art, for example as described inEP-A-1 630 173.

The refolded G-CSF can be prepared subsequent to refolding and previousto the first chromatographic step, for example by means of filtration,concentration, precipitation, acidification and/or dialysis.

In many cases it will be advantageous to purify the folding setupprevious to the first chromatographic step, i.e. to removehigh-molecular particles, which are mostly protein aggregates that havebeen formed in folding. Said purification can be conducted by means ofdepth filtration, wherein a granulate bulk material serves as filtermeans. The solid particles are larger than the pores of the filter meansor are held back by absorption at the inner surface of the bulk.

In depth filtration, the use of cellulose ester fibers as filter meansis preferred. Suitable filter means as well as correspondinginstructions for use are, for example, available from Millipore underthe trade names Millistak Plus C0HC and Millistak+B1HC.

Preferably, the folding setup is acidified previous to the depthfiltration, so that the filtrate can be immediately employed for thecation exchange chromatography in a particularly efficient manner.Herein, the ph value of the folding setup is preferably set to below4.0, particularly preferably to 3.2.

For the cation exchange chromatography, conventional commerciallyavailable matrices can be employed. Herein, the G-CSF binds to thecation exchange matrix within a specific pH range due to its positivetotal charge, while most of the contaminating substances like nucleicacids, lipopolysaccharides and proteins originating from host cells aswell as ionic isomers of G-CSF and altered forms of G-CSF havingdifferent pH values are not capable of binding or of being removed bymeans of washing.

Suitable cation exchange matrices include, but are not limited to,carboxymethyl (CM) cellulose, AG 50 W, Bio-Rex 70, carboxymethyl (CM)Sephadex, sulfopropyl (SP) Sephadex, carboxymethyl (CM) sepharose CL-6B,CM sepharose HP, Hyper D-S ceramic (Biosepra) and sulfonate (S)sepharose, SP sepharose FF, SP sepharose HP, SP sepharose XL, CMsepharose FF, TSK gel SP 5PW, TSK gel SP-5PW-HR, Toyopearl SP-650M,Toyopearl SP-650S, Toyopearl SP-650C, Toyopearl CM-650M, ToyopearlCM-650S, Macro-Prep High S Support, Macro-Prep S Support, Macro-Prep CMSupport etc.

Suitable matrices and protocols for conducting the cation exchangechromatography can be taken from the product information of supplierslike Amersham Biosciences (http://www.amershambiosciences.com, now GEHealthcare) or Bio-Rad (http://www.bio-rad.com) by the person skilled inthe art.

Sulfopropyl matrices, in particular the products SP Sepharose XL and SPSepharose FF (Fast Flow), available by Amersham Biosciences, Freiburg,Germany (now GE Healthcare), are preferably used as matrix for thecation exchange chromatography.

In a preferred embodiment of the present invention, in which two cationexchange chromatographies are conducted, namely before and after thehydrophobic interaction chromatography, respectively, a sulfopropylmatrix is employed in both cases, particularly preferably SP SepharoseXL in the first cation exchange chromatography and SP Sepharose FF inthe second cation exchange chromatography.

Suitable buffers for the cation exchange chromatography include maleate,malonate, citrate, lactate, acetate, phosphate, HEPES and Bicin buffers.Preferably, the concentration of the buffer lies between 10 and 100 mM,preferably between 20 mM and 50 mM. For purifying the G-CSF, the pHvalue of the buffer should possibly not be higher than 7.0, preferablynot higher than 6.5.

In a preferred embodiment, 20 mM sodium acetate, pH 5.0, which isemployed for equilibrating and washing, is used for the cation exchangechromatography.

In case a second cation exchange chromatography is conducted, 50 mMsodium phosphate, pH 5.4, is herein preferably used for equilibratingand washing.

Subsequently to washing, the G-CSF can be eluted from the column bymeans of an alteration, in case of the cation exchange chromatography bymeans of an increase in pH value or an increase in ionic strength.

Preferably, the elution is effected by means of increasing the ionicstrength. In case 20 mM sodium acetate, pH 5.0, is used as buffer, asolution of 20 mM sodium acetate, pH 5.0, and 200 mM NaCl is, forexample, suitable for the elution.

Further suitable conditions for the cation exchange chromatography canbe taken from the relevant literature, like for example from the manual“Ion Exchange Chromatography—Principles and Methods” by AmershamBiosciences, Freiburg, Germany (now GE Healthcare), 2002.

The salt concentration in the charging buffer for the cation exchangechromatography should be sufficiently low in order to allow binding tothe matrix, wherein binding also depends on the pH value of thesolution.

Within the scope of the cation exchange chromatography, differentbuffers can be employed for charging and binding to the matrix, forexample buffers selected from the group consisting of acetate, citrate,Tris/HCl, Tris/acetate, phosphate, succinate, malonate,2-(N-morpholinoethanesulfonate) (MES) and other buffers.

After charging the column, the column is washed and subsequently theproteins are eluted from the column. Herein, the elution can beconducted by means of increasing the ionic strength, which is effectedby means of increasing the salt concentration in the buffer solution.Alternatively, an increase in pH value is suitable. Herein,discontinuous step gradients, linear gradients or a suitable combinationof such gradients can be employed.

Elution buffers suitable for washing and for the elution can be selectedfrom acetate, citrate, Tris/HCl, Tris/acetate, phosphate, succinate,malonate, MES and other suitable buffers with the addition of salts likeNaCl or KCl. The ionic strength and the salt concentration, by means ofwhich the elution is achieved, are dependent on the pH value of thebuffer solution. The higher the pH value of the buffer, the lower is theionic strength that is required for the elution of the proteins from thecolumn.

The hydrophobic interaction chromatography can also be conducted withconventional matrices. Suitable are matrices like butyl, phenyl or octylsepharose (Amersham Biosciences, now GE Healthcare), Makro-Prep-methylor t-butyl (Bio-Rad) and Fractogel EMD with propyl or phenyl ligands(Merck).

Preferably, the hydrophobic ligands are butyl, phenyl or octyl groups,particularly preferably they are phenyl groups. Herein, the products byAmersham Biosciences (now GE Healthcare) can be employed.

Suitable matrices and protocols for conducting the hydrophobicinteraction chromatography can be taken from the product information ofsuppliers like Amersham Biosciences (http://www.amershambiosciences.com,now GE Healthcare) or Bio-Rad (http://www.bio-rad.com) by the personskilled in the art.

Preferably, the matrix is Phenyl Sepharose HP (High Performance),available by Amersham Biosciences (now GE Healthcare).

Conventional buffers, which are also employed in other types ofchromatography, are suitable as buffers for the hydrophobic interactionchromatography. In a preferred embodiment, a citrate buffer is used.Advantageously, the elution is conducted by means of increasing the pHvalue. A pH gradient from about pH 3.0 to about 6.0 has proven to beparticularly suitable.

Further conditions suitable for the hydrophobic interactionchromatography can be taken from the relevant literature, like forexample from the manual “Hydrophobic InteractionChromatography—Principles and Methods” by Amersham Biosciences (now GEHealthcare), Freiburg, Germany, 2002.

In general, the person skilled in the art is familiar with thechromatographic principles utilized in the method according to thepresent invention; in any case, they are described in detail inestablished manuals or protocols by the suppliers of chromatographymatrices, columns and other means.

The tangential flow filtration (TFF), which is conducted within thescope of one embodiment of the present invention subsequently to thechromatic purification, in particular subsequently to the only or thelast cation exchange chromatography, can be conducted by means ofconventional TFF systems and protocols, like for example supplied by thecompanies Millipore and Pall Corporation. The TFF is a filtration as anadditional purification step in contrast to the previous purificationsteps of the cation exchange chromatography and the hydrophobicinteraction chromatography.

The G-CSF purified within the scope of the present invention isexpressed in host cells by means of conventional gene-technologicalmethods. Preferably, it is human G-CSF. Various expression systems forthe expression in E. coli cells are commercially available. Suitable is,for example, the expression of human G-CSF under the control of aninducible promoter, for example an IPTG-inducible promoter, see forexample Sambrook and Russel, Molecular Cloning—A Laboratory Manual,3^(rd) edition 2001, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA, chapter 15, or established manufacturers' protocols,for example by Proinega or Stratagene.

Fermentation is conducted according to standard protocols, like they aredescribed in patent and scientific literature, for example in a two-stepprocess consisting of a batch cultivation and a fed batch cultivation.

Harvesting the so-called inclusion bodies containing the G-CSFoverexpressed in E. coli and the lysis of said inclusion bodies havepartly been described in the patent literature discussed in the above.However, suitable protocols can also be found in standard works onprotein chemistry as well as in laboratory manuals. The same applies forsolubilizing and refolding, which are objects of various patentdocuments, as has been discussed in the above.

The invention also relates to pharmaceutical preparations containing theG-CSF obtained according to the present invention. The G-CSF obtainedcan either be stored in the form of a lyophilisate or in liquid form. Itis administered either subcutaneously or intravenously. Suitableadjuvants in the formulations of the recombinantly expressed G-CSF are,for example, stabilizers like sugar and sugar alcohols, amino acids andtensides like for example polysorbate 20/80 as well as suitable buffersubstances. Examples for formulations are described in EP 0 674 525, EP0 373 679 and EP 0 306 824, see also the trade products Neupogen® andGranocyte in the “ROTE LISTE 2004”.

EXAMPLES

The following examples are intended to illustrate the present inventionwithout limiting the scope thereof.

The human G-CSF was expressed under the control of an IPTG-induciblepromoter in E. coli cells. Examples for suitable expression systems canbe taken, for example, from the laboratory manual Sambrook and Russell,Molecular Cloning—A Laboratory Manual, 3^(rd) edition, 2001, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA, chapter 15, orfrom established manufacturers' protocols, for example by Promega orStratagene.

The fermentation was conducted according to standard protocols, likethey are described in patent and scientific literature, in a two-stepprocess comprising a batch cultivation and a fed batch cultivation. Thebacteria were cultivated for 17 to 18 hours before they were stimulatedby means of adding 1 mM IPTG to form the recombinant G-CSF. Theinduction period was 4.0 hours.

Harvesting the bacteria was conducted by means of beaker centrifugationfor 20 min at 5,000 g and 4° C. After the centrifugation, thesupernatant was discarded and the cells were filled up again with buffer(20 mM sodium phosphate, pH 7.0; 1 mM EDTA) to the fermentation volumebefore they were lysed by means of three passages at 800 bar.Subsequently, the lysate was purified by means of separation(CSA1-Separator, Westfalia, Oelde, Germany).

Concentrating the Inclusion Bodies

In principle, the suspension of the inclusion bodies that has beenobtained by harvesting can immediately be used for the subsequentsolubilization. In this case, however, the maximum achievable proteinconcentration in the solubilisate is strongly limited, which may lead tolimitations during folding. Thus, the suspension of the inclusion bodiesshould be concentrated by means of centrifugation subsequently toharvesting and washing in order to achieve a high protein concentrationin the solubilisate.

The suspension of the inclusion bodies was centrifuged for 20 minutes at10,000 g in a beaker centrifuge. The paste of inclusion bodies that isobtained by means of centrifugation can be stored at −20° C. for atleast 12 weeks.

Solubilization

In order to allow effective solubilization of larger pellets ofinclusion bodies in a relatively short time, mechanical crushing of saidpellets, for example by means of Ultra Turrax treatment, is furthermorerequired. The paste of the inclusion bodies that was obtained by meansof centrifugation was weighed, mixed with 9.0 ml solubilizing buffer (30mM Tris, 1 mM EDTA, 6.0 M guanidine-HCl, 100 mM GSH, pH 8.0) per graminclusion bodies and crushed by means of Ultra Turrax treatment. Thesetup was thoroughly vortexed and was then incubated on a roller mixeror a magnetic stirrer at room temperature for about 2 hours.

Refolding

The protein concentration in the solubilisate was determined by means ofthe method according to Bradford using BSA as standard protein. Forfolding, the amount necessary to achieve a protein concentration of 700μg/ml in a desired amount of buffer was added to the refolding buffer(30 mM Tris, 2 mM GSSG, 2 mM GSH, 3 M urea, pH 7.5; 4° C.). Thecorresponding amount of solubilisate was added slowly and steadily whilestirring with a magnetic stirrer in order to avoid locally increasedconcentrations of solubilisate or protein. The afflux speed and the modeof mixing can be adjusted to the respectively employed volume ofsolubilizing setup. After the addition of the solubilisate had beencompleted, the setup was incubated for at least 12 hours at 4° C. Duringthis period no further mixing was required.

Depth Filtration

Subsequently to refolding, the refolding setup is filtrated before thefirst chromatographic step is conducted. Herein, for example, a depthfilter can be used for the filtration, for example a suitable filter byMillipore, Schwalbach, Germany. Previously to the filtration, the pHvalue is adjusted to pH 3.2 by means of 2 M citric acid.

Conducting the First Cation Exchange Chromatography

The first chromatographic step serves for capturing the target proteinand separates refolding agents like urea, GSH, GSSG, as far as those arepresent in the folding setup, from the target protein. In said step,incorrectly folded protein species and host cell proteins are alsoseparated. Here, a cation exchange chromatography is employed. SPSepharose XL by Amersham Biosciences (now GE Healthcare) is used asmatrix. The chromatography is conducted at pH 5.0.

The SP Sepharose XL matrix was equilibrated with 1.5 column volumes of20 mM sodium acetate, pH 5.0. The filtered refolding setup was loadedonto the column and was subsequently washed with 1.5 column volumeswashing buffer (20 mM sodium acetate, pH 5.0). Subsequently, the G-CSFwas eluted from the column with 3 column volumes elution buffer (20 mMsodium acetate, 200 mM NaCl, pH 5.0). The purity of the eluted G-CSF wasdetermined by means of rpHPLC; it was higher than 80%. As related to thefiltered folding setup, the yield was also higher than 80%.

Conducting the Hydrophobic Interaction Chromatography

In the second chromatographic step, a further purification of the G-CSFis conducted on the basis of the eluate of the SP Sepharose XL. Inparticular the product-related contaminations are substantiallydepleted. Here, a hydrophobic interaction chromatography with PhenylSepharose HP by Amersham Biosciences (now GE Healthcare) is conducted.

The Phenyl Sepharose HP column was first equilibrated with 2 columnvolumes of 12% buffer B, 88% buffer A (buffer B: 20 mM sodium citrate,pH 6.7; buffer A: 20 mM sodium citrate, pH 2.7, 110 mM NaCl). Then, theeluate of the SP Sepharose XL column, which had previously been dilutedwith 5 volumes of buffer A (20 mM sodium citrate, pH 2.7, 110 mM NaCl),was applied onto the column. Subsequently, the column was washed with 2column volumes of 12% buffer 13, 88% buffer A and a linear gradient from12% to 90% buffer B was run in 5-8 column volumes. The elution occurredwithin the scope of said linear pH gradient from about pH 3.0 to about6.0. Finally, the column was rinsed with 3 column volumes of 90% bufferB, 10% buffer A.

The elution fractions were tested for their purity by means of rpHPLCand fractions having a purity higher than 95% were combined.

The G-CSF obtained after the hydrophobic interaction chromatography hada purity of more than 96%. The yield from the HIC step was almost 80%.

Conducting the Second Cation Exchange Chromatography

In the third chromatographic step, a further purification of the G-CSFto a purity of more than 99% is conducted on the basis of the eluate ofthe Phenyl Sepharose HP. In particular the product-relatedcontaminations are substantially depleted. Here, a cation exchangechromatography is again employed. The SP Sepharose FF by AmershamBiosciences (now GE Healthcare) is used herein.

The SP Sepharose FF column was equilibrated with 3 column volumes of100% buffer A (50 mM sodium phosphate, pH 5.4). Subsequently, the eluateof the hydrophobic interaction chromatography was applied onto thecolumn and the column was rinsed with 2 column volumes of 100% buffer A(50 mM sodium phosphate, pH 5.4). Herein, the sample that was appliedcontained about 60 mM NaCl and had a pH value of 4.0-4.2. The elutionwas conducted by means of a combination of step and linear pH gradient.The first step ran up to 10% buffer B (50 mM sodium phosphate, pH 6.4)and maintained said concentration for 1.5 column volumes. This wasfollowed by a gradient over 1 column volume from 10 to 15% buffer B (50mM sodium phosphate, pH 6.4). G-CSF was eluted in a linear gradient from15% to 35% buffer B (50 mM sodium phosphate, pH 6.4) over 12.5 columnvolumes, wherein collecting the eluate with increasing absorption wasconducted at 280 mm. Finally, the column was rinsed in one step to 100%buffer B (50 mM sodium phosphate, pH 6.4).

The elution fractions were tested for their purity by means of rpHPLCand fractions having a purity of more than 99% were combined. The totalyield was 80%.

The Met-G-CSF that was obtained as a result of the chromatographic stepsdescribed in the above had a purity of at least 99.5% after all HPLCanalyses (rp, SEC and IEX).

Determining the contaminations also resulted in a very strong depletionof DNA, endotoxin and host cell protein.

Determining the Biological Activity

The activity of the G-CSF obtained by the method according to thepresent invention was determined by means of a bioassay and was comparedto the activity of a standard, commercially available G-CSF (Neupogen®).To this end, the mouse cell line NFS-60 was used, which is responsive toG-CSF. Said cell line was cultivated in RPMI 1640 medium (Bachem,Heidelberg, Germany), which contained 1.5 g/l sodium carbonate, 4.5 g/lglucose, 10 mM Hepes and 1.0 mM sodium pyruvate and had beensupplemented with 2 mM glutamine, 10% FCS, 0.05 mM 2-mercaptoethanol and60 ng/ml G-CSF.

For the activity test, the cells were washed twice with medium withoutG-CSF, placed in 96-well plates at a concentration of 2×10⁴ cells perwell and were incubated for three days at 37° C. and 4.5% CO₂ withvarying concentrations of the purified G-CSF and the standard.Subsequently, the cells were stained with XTT reagent and the absorptionat 450 nm was measured in a microtiter plate reader. It showed that thecells that had been treated with the G-CSF purified according to thepresent invention grew just as well as those cells that had been treatedwith the standard, which led to the conclusion that both G-CSF sampleshad the same biological activity.

In the gel electrophoretic analyses (SDS-PAGE, Western Blot, isoelectricfocusing), the Met-G-CSF obtained after the chromatographic purificationalso behaved like the Neupogen® used as standard.

In order to determine the folding yield, an rpHPLC, wherein the proteinis denatured, may be conducted subsequently to obtaining the G-CSF. Dueto maintained disulfide bridges, different disulfide-bridged speciesoften have different hydrophobic surfaces and can therewith be separatedin the rpHPLC. Thus, only the detection of the correct disulfide bridgecan be conducted with said method. The former, however, is a decisiveand for many small and correspondingly bridged proteins an alreadysufficient criterion for correct folding. A size exclusion (SE)-HPLC canalso be conducted as a further analysis method.

Suitable materials and protocols for conducting the rpHPLC or theSE-HPLC can be taken from the product information by suppliers likeVydac (http://www.vydac.com) or TOSOH Bioscience(http:l/www.tosohbiosep.de) by the person skilled in the art.Determining the yield of Met-G-CSF is also described in Herman et al,(1996) Pharm. Biotechnol. 9: 303-328). Herein, the exact proportion ofMet-G-CSF is determined by means of integration of the peak area andconversion on the basis of the extinction coefficient.

Subsequently to purification, the G-CSF can be analyzed with respect toits amount and its activity. A qualitative analysis can be conducted viaan SDS-PAGE analysis with subsequent Coomassie Brilliant Blue Stainingor an rpHPLC. A commercially available G-CSF preparation can be used asstandard for the analyses. In addition, a peptide map or a massspectroscopy can be conducted. The activity of the G-CSF purified can bedetermined by means of different biological test methods, like they are,for example, described in Shirafuji et al. (1989) Exp. Hematol. 17 (2):116-119; Oh-Eda et al. (1990) J. Biol. Chem. 265 (20): 11432-11435;Stute et al. (1992) Blood 79 (11): 2849-2854 and Oshima et al. (2000)Biochem. Biophys. Res. Commun. 267 (3): 924-927.

Incidentally, all chromatographies are conducted according to therecommendations and protocols of the suppliers of the matrices or thecolumns (for example with respect to flow rate, column volumes employedfor washing or for elution, diameters and bed heights of the columns,etc.).

1-12. (canceled)
 13. A method for purifying recombinantgranulocyte-colony stimulating factor (G-CSF), comprising at least onecation exchange chromatographic step and at least one hydrophobicinteraction chromatographic step, wherein said chromatographic steps areimmediately consecutive in optional order.
 14. The method of claim 13,further comprising a tangential flow filtration step subsequent to thelast cation exchange chromatographic step.
 15. The method of claim 13,comprising two cation exchange chromatographic steps which are conductedbefore and after the hydrophobic interaction chromatographic step,respectively.
 16. The method of claim 15, further comprising atangential flow filtration step subsequent to the last cation exchangechromatographic step.
 17. The method of claim 13, wherein no anionexchange chromatographic step is conducted.
 18. The method of claim 13,wherein no gel filtration step is conducted.
 19. The method of claim 13,wherein no HPLC step is conducted.
 20. The method of claim 13, whereinno affinity chromatographic step is conducted.
 21. The method of claim13, wherein no hydroxyapatite chromatographic step is conducted.
 22. Themethod of claim 13, wherein a sulfopropyl matrix is used for the cationexchange chromatographic step.
 23. The method of claim 13, whereinphenyl groups are used as hydrophobic ligands for the hydrophobicinteraction chromatographic step.
 24. A method for producing apharmaceutical preparation containing recombinant G-CSF andpharmaceutically acceptable adjuvants, comprising purifying C-CSFaccording to claim 13 and combining said purified G-CSF with apharmaceutically acceptable adjuvant.
 25. The method of claim 24,wherein the pharmaceutically acceptable adjuvant is selected from thegroup consisting of like buffers, salts and stabilizers.